Vitamin B12 or cobalamin is a reactive and scarce, yet essential cofactor that is acquired by mammals from the diet. Studies on patients with inborn errors of cobalamin metabolism have led to the discovery of nine genes involved in the tailoring, chaperoning, and utilization of cobalamin. Of these, two, CblA and adenosyltransferase (ATR), are involved in the mitochondrial trafficking pathway and support the function of B12-dependent methylmalonyl-CoA mutase (MCM). Mutations in CblA, ATR or MCM lead to methylmalonic aciduria, a devastating metabolic disorder that often results in infant mortality. Extensive biochemical studies in our laboratory on the bacterial orthologs of human MCM and its G-protein chaperone, CblA, have demonstrated that the G-protein gates delivery of the B12 cofactor, 5-deoxyadenosylcobalamin (AdoCbl), from ATR to MCM. The G-protein forms a high affinity complex with MCM, and protects the cofactor against inactivation during MCM catalysis. It also ejects inactive cofactor when it is formed accidentally during the radical rearrangement reaction catalyzed by MCM. While the bacterial proteins have been useful models, significant structural and functional differences between them and the human proteins suggest limitations for their continued use for understanding the structural basis of catalysis and allosteric communication between CblA and human MCM, and how these might be corrupted by patient mutations. With the availability of human CblA, MCM and ATR in amounts needed for biophysical studies, we are now positioned to approach their study directly. We will elucidate the structural enzymology of the human mitochondrial B12 trafficking pathway by completing the following two specific aims. (i) We will characterize the energetics, stoichiometry and modulation of complex formation between MCM and CblA by nucleotides (GDP and GTP) and by AdoCbl, the catalytic activities (GTPase and isomerase) of the two proteins within the complex, and the chaperone functions of CblA, including gating of AdoCbl transfer from ATR to MCM. These studies will set the foundation for interrogating the deficits associated with select missense mutations in MCM described in methylmalonic aciduria patients. (ii) We will structurally characterize the bacterial and human MCM-G-protein complexes using single particle electron microscopy and use the available crystal structures of the individual proteins to construct 3D models of the complexes. We will also use X-ray crystallography to obtain the crystal structure of the bacterial MCM-G-protein complex. These studies will provide fundamental insights into the structure of, catalysis by, and bidirectional allosteric communication within the MCM-G-protein complex and generate clinically relevant information on how patient mutations impact these functions at a molecular level.